Sponge-derived matter is assimilated by coral holobionts

Coral reef biodiversity is maintained by a complex network of nutrient recycling among organisms. Sponges assimilate nutrients produced by other organisms like coral and algae, releasing them as particulate and dissolved matter, but to date, only a single trophic link between sponge-derived dissolved matter and a macroalgae has been identified. We sought to determine if sponge-coral nutrient exchange is reciprocal using a stable isotope ‘pulse-chase’ experiment to trace the uptake of 13C and 15N sponge-derived matter by the coral holobiont for three coral species (Acropora cervicornis, Orbicella faveolata, and Eunicea flexuosa). Coral holobionts incorporated 2.3–26.8x more 15N than 13C from sponge-derived matter and A. cervicornis incorporated more of both C and N than the other corals. Differential isotopic incorporation among coral species aligns with their ecophysiological characteristics (e.g., morphology, Symbiodiniaceae density). Our results elucidate a recycling pathway on coral reefs that has implications for improving coral aquaculture and management approaches.


Coral and Symbiodiniaceae Separations
To prepare for downstream analyses, the coral fractions, host and symbiotic micro-algae (family Symbiodiniaceae), were manually separated.Symbiodiniaceae encompasses a diverse set of unicellular dinoflagellates 1 and corals are largely associated with those belonging to seven genera 2 .We did not genotype the dinoflagellates recovered in this study, so the terms 'symbiotic micro-algae' and 'Symbiodiniaceae' are used to refer to any species of dinoflagellate that separated out during this process.Scleractinian fragments were thawed and airbrushed with an aerosolized jet of 0.22 µm filtered seawater to physically separate the coral tissue and skeleton and suspend the coral tissue material into a homogenate.To separate host tissue from Symbiodiniaceae cells, the homogenate was centrifuged at 2000 x g for 3-5 minutes.Centrifugation formed a pellet comprised of Symbiodiniaceae cells and a homogenate of host material.The host homogenate was pipetted into a separate sterile 50 ml Falcon tube.The homogenate, Symbiodiniaceae pellet, and skeletal fragments were frozen and transported to Appalachian State University where they were stored at -20 C until further processing.At Appalachian State, host homogenates and Symbiodiniaceae pellets were thawed and checked for purity.Impure fractions were recombined, homogenized with a tissue homogenizer (maximum speed for ~15 sec) to physically separate Symbiodiniaceae cells from host tissue, and centrifuged (3000 x g, 6 min.) to pellet the Symbiodiniaceae cells.Following centrifuging, fractions were checked for purity under the microscope, and the process was repeated until at least 80% purity was reached.
E. flexuosa fractions were separated using a different process.First, the frozen coral fragments were lyophilized (Labconco™ FreeZone™ Bulk Tray Dryer) for 22-24 hours, until they were completely dry.Following lyopholization, the axial skeleton was removed, and the tissue was ground up using a mortar and pestle.Separate mortar and pestle sets were used for control and enriched samples.The ground tissue was weighed and then rehydrated in 10 ml of MilliQ water in a sterile 15 ml Falcon tube.Very quickly following rehydration, the sclerites (skeletal fragments) sank to the bottom of the tube and the remaining host homogenate was pipetted into a new tube taking care not to transfer the sclerites.The host homogenate was homogenized further using a tissue homogenizer for ~15 sec at maximum speed and centrifuged at 4000 x g for 5 minutes to separate the fractions.The centrifugation step was repeated as necessary until the host homogenate and Symbiodiniaceae pellets were at least 80% pure.
Following fraction separations, 50 ml of the Symbiodiniaceae fraction from each sample was transferred to a cryovial with 50 ml of 10% paraformaldehyde (PFA) to fix the cells for Symbiodiniaceae density estimates.Fixed Symbiodiniaceae samples were stored in the 4°C refrigerator.The pure host homogenates and remaining Symbiodiniaceae pellets were stored in the -20°C freezer.

Processing of Enriched Water Samples
During the 'pulse-chase' experiment, six 500 ml enriched water samples (n=3 from the 'pulse' and n=3 from a subset of the sponge-containing aquaria ~1.5 hrs into the 'chase') were collected in acid-washed polycarbonate bottles for bulk dissolved organic carbon (DOC) and total nitrogen (TN) analysis.Note that the tank water used for the 'pulse' was 0.22 µm filtered and the labeled bicarbonate provided is inorganic, thus, the DOC was composed of both background from the seawater and sponge-derived DOC.For the 'chase' samples, the water in the aquaria was not pre-filtered, so DOC sources include tank seawater, sponge-and coralderived DOC, and possibly a small contribution from microbial production.TN includes both organic and inorganic nitrogen compounds, and for the 'pulse' was composed of background nitrogen, the labeled inorganic compounds, and sponge-derived nitrogenous dissolved matter (NDM).For the 'chase' the labeled compounds are replaced with possible microbe-and coralproduced NDM, though we expect this contribution was minimal.
Each enriched water sample was individually passed through a polytetrafluoroethylene (PTFE) 0.22 µm, 47 mm filter (Omnipore, EMD Millipore Corporation, Billerica, MA, USA) encased in an acid-washed in-line perfluoroalkoxy alkane (PFA) filter holder (Advantec, Cole-Parmer, Vernon Hills, IL, USA) using peristalsis (MasterFlex L/S pump and pump heads, Cole-Parmer, Vernon Hills, IL, USA).All filter tubing (Masterflex® L/S® Precision Pump Tubing, PharMed® BPT, L/S 25, Radnor, PA, USA) was acid-washed.Filtering rates were kept slow to avoid the bursting of microbial cells on the filter membrane which could cause sample contamination.Subsamples of the filtrate (25 ml) were collected in acid-washed, combusted 40ml amber EPA vials and acidified to pH 3 with 12M trace metal grade hydrochloric acid (HCL, OptimaTM, Fisher Chemical, Fisher Scientific, Hampton, NH, USA).EPA vials were wrapped in tinfoil to limit light exposure and stored at 4 ºC.The acidified samples were sent to Woods Hole Oceanographic Institute for dissolved organic carbon (DOC) and total dissolved nitrogen (TN) analysis using a Shimadzu TOC-VCSHTOC analyzer 3 .

Stable Isotope Analysis Equations
Carbon and nitrogen stable isotope ratios are expressed in standard delta notation and calculated using the equation from Fry 4 as follows: where Rsample is the ratio of 13 C/ 12 C or 15 N/ 14 N in the sample material (sponge, coral, or Symbiodiniaceae), and Rstandard is the known ratio of the isotopes in a standard reference material (Vienna Pee Dee Belemnite (VPDB) for C; Rstandard = 0.011180, and for N; Rstandard= 0.0036765).The final multiplication by 1000 is meant to amplify any small differences between samples and reference values.Values for δ are reported in permil units ( 0 /00), and high δ values indicate enrichment of the heavy isotope while negative δ value indicate a sample depleted in heavy isotopes relative to the standard material.
Then, following the process outlined in Fry 4 , we used the δ values to calculate fractional abundance (F; i.e., fractions of total C and N), which is preferred over δ for downstream calculations of enriched samples as it generates less errors.We start by rearranging the definition of δ as: where δ is the δ 13 C or δ 15 N for the sample and the corresponding C or N Rstandard is used.
We then use F to calculate excess fractional abundance for an enriched sample (Esample) as the difference between the fractional abundance of the enriched sample (Fsample) and the average fractional abundance of the background samples (Fbkgd), where background samples are all T0 (i.e., initial) samples for each species and fraction.
Next, we used slightly modified versions of the equations put forth in Rix et al. 5 to calculate total 13 C or 15 N incorporation and incorporation rate.To calculate total incorporation (I) we multiplied the excess fractional abundance (Esample) by the total C or N content (µmol) of the sample (Asample):  =  )*+,-.*  )*+,-. .Lastly, we normalized the 13 C or 15 N incorporation (I) of each sample to its total C or N content (Asample) and the number of hours the sample was exposed to the isotope (T) during the 'chase' (3 or 6 hrs) to obtain a biomass-specific incorporation rate of heavy isotope (IRTracer) into the tissue of each sample.
IRTracer is reported in the text results, Fig. 3, and Supplementary Table S4 as 'Incorporation Rate (µmol of 13 C or 15 N mmol of C or Ncoral or Symbiodiniaceae -1 hr -1 ).' Values of δ 13 C, δ 15 N, total carbon and total nitrogen content (µmol of C or N) used in the above calculation are publicly available at https://www.bco-dmo.org/dataset/889857 6.

Coral Surface Area Measurements
Surface area was calculated for coral fragments following two well-documented methods: Image J measurements and the aluminum foil method 7 .ImageJ coral surface area measurements were completed utilizing planar photography and ImageJ software 8 .For A. cervicornis and O. faveolata, fragment surface area was measured using the frozen, airbrushed skeleton for each sample, while for E. flexuosa, the entire fragment, prior to lypopholization as detailed above, was used.To obtain images of A. cervicornis and E. flexuosa, the fragments were held at an upright position, similar to their natural growth direction, and photographed from four sides (rotated 90°).O. faveolata are dome-shaped mounding corals, so photographs were only taken from above.A ruler was held in alignment with the fragments for scaling purposes.Photographs were individually uploaded to ImageJ and pixel dimensions were set using the straight-line tool and 'set scale' option.Using the polygon tool to drag an outline around the perimeter of the fragment, the enclosed area was calculated with the 'measure' function (in cm 2 ).The area of all four sides was summed to estimate the surface area of the skeletal fragment.
The aluminum foil method was completed based on published methods 7 , but briefly, small pieces of aluminum foil were cut and carefully measured (cm 2 ) and weighed (g) to obtain a standard weight per unit of area (g/cm 2 ) for the foil.The foil used in this study had a standard weight per unit of area of 0.00618 g/cm 2 .Coral fragments were carefully covered with aluminum foil and all excess foil was trimmed until there was no overlap.Each foil wrapping was carefully removed from the fragment, and weighed.The fragment surface area was calculated using the standard weight per unit area of the aluminum foil (Surface area of coral fragment = mass of coral fragment foil*0.006180525794g/cm 2 ).All surface measurement data is publicly available at https://www.bco-dmo.org/dataset/880711 9.

Symbiodiniaceae Density
Symbiodiniaceae density (cells/cm 2 ) was estimated for each coral fragment using the following procedure.First, 20 µl of the PFA-fixed Symbiodiniaceae cells from each sample were stained with either 2 µl (E.flexuosa) or 5 µl (A.cervicornis and O. faveolata) of Trypan Blue to increase their visibility under the microscope.If the cells were too numerous to be counted accurately, they were further diluted with 175 µl of MilliQ water.A subsample (10 µl) of the dilution was then placed into the cell counting chamber of a hemocytometer (Marienfeld Superior Neubauer Improved Chamber) and cells were counted in 4 quadrants following the standard Neubauer protocol as suggested by Electron Microscopy Sciences (https://www.emsdiasum.com/microscopy/technical/datasheet/68052-14.aspx).Cell counts from all quadrants were summed.Symbiodiniaceae abundance in the dilution (cells/ml dilution) for each fragment was obtained by multiplying the total cell count by the dilution factor and the standard cell abundance for the hemocytometer (10,000 cells/ml per manufacturer) and dividing by the number of quadrants counted (4).

𝑺𝒚𝒎𝒃𝒊𝒐𝒅𝒊𝒏𝒊𝒂𝒄𝒆𝒂𝒆 𝑨𝒃𝒖𝒏𝒅𝒂𝒏𝒄𝒆 (𝒄𝒆𝒍𝒍𝒔/𝒎𝒍) = 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐴𝑏𝑢𝑛𝑑𝑎𝑛𝑐𝑒 * 𝑉𝑜𝑙𝑢𝑚𝑒 ℎ𝑜𝑠𝑡 ℎ𝑜𝑚𝑜𝑔𝑒𝑛𝑎𝑡𝑒
Finally, the Symbiodiniaceae density for each fragment was calculated by standardizing the Symbiodiniaceae abundance to the total surface area of the fragment (cm 2 ) as determined by the ImageJ method detailed above.

𝑺𝒚𝒎𝒃𝒊𝒐𝒅𝒊𝒏𝒊𝒂𝒄𝒆𝒂𝒆 𝑫𝒆𝒏𝒔𝒊𝒕𝒚 (𝒄𝒆𝒍𝒍/𝒄𝒎
All data associated with the calculation of Symbiodiniaceae densities in the coral fragments is publicly available at https://www.bco-dmo.org/dataset/880711 9.S1.Total counts of sponge, coral and Symbiodiniaceae samples included at each point during the 'pulse-chase' experiment.Black cells indicate that no samples were collected at that time point.Note: There were a total of 8 experimental tanks (3 control, 5 enriched), but several coral and Symbiodiniaceae samples with abnormal 13 C or 15 N values (high or low depending on expected values for each treatment) were removed in an abundance of caution and this is reflected in the 'chase' sample counts.9 Supplementary Table S2.Mean (± SD) δ 13 C and δ 15 N for sponge, coral, and Symbiodiniaceae tissues throughout the 6-hr 'pulsechase' experiment.'Pulse' column values are from sponge samples collected at the end of the 3-hr 'pulse.'T0, indicates the initial samples taken prior to the start of the experiment, while T3, and T6 denote the two sampling times at the mid-point and the end of the 'chase,' respectively.Labeled isotopes provided to sponges via dissolved organic bicarbonate ( 13 C), ammonia and nitrate ( 15 N).

Supplementary Table
Bolded red text in the "Change in δ 13 C" and "Change in δ 15 N" columns denotes a net loss of the heavy isotope during the 'chase,' while bolded blue text denotes a net gain of the heavy isotope during the 'chase.'Note that releases and gains are not necessarily significant.'ND' is used to denote an experimental time point where samples were not collected for the indicated group.Sample groups without a standard deviation did not contain more than one individual.Supplementary Table S3.Total incorporation (µmol) and incorporation rates (heavy isotope (µmol)/total isotope in tissue (mmol) per hour) of 13 C and 15 N in sponge-derived dissolved matter by coral and Symbiodiniaceae during the 6-hr 'chase.'Data is given for both sampling time points (T3 and T6).Bolded blue text in T6 columns denotes a value that is higher than its T3 counterpart, conversely, bolded red text denotes a T6 value that is lower than its T3 counterpart.For total incorporation a blue value at T6 would suggest a net gain of the heavy isotope in the tissue, and a red value a loss of the heavy isotope, between hours 3-6 of the 'chase.'For incorporation rate, a red value in T6 indicates that the heavy isotope was assimilated into the coral or Symbiodiniaceae tissue faster in the first half of the 'chase,' while a blue value indicates a faster rate in the last half of the 'chase.'A bolded black value in either column indicates no difference across 'chase' time points.Note differences are not necessarily significant.

Species and Fraction
Carbon (

Pairwise PERMANOVA Test Results
Here we report the results from the post-hoc pairwise tests associated with the PERMANOVA tests reported in Supplementary Table S4.All post-hoc tests were completed using the 'pairwiseadonis' package in R with 999 permutations and Bonferroni corrections.Only pairwise results that are reported in text are included here.For access to unreported pairwise tests please contact the corresponding author.Asterisks denote significant (p<0.05)differences.

Table S4 .
PERMANOVA results of multivariate tests reported in the text.Significance was obtained from p-values calculated by permutations of the residuals under a reduced model using 999 unique permutations.Associated post-hoc pairwise PERMANOVAS were completed with Bonferroni corrections and are detailed in the Supplementary Results (pgs.18-21).Asterisks denote significant differences (p<0.05).

continued on pg. 12 Supplementary Table S4 continued from pg. 11
s.